Molecular pathogenesis and protective approaches in Burkholderia pseudomallei infection .

1. Molecular pathogenesis and protective approaches in Burkholderia pseudomallei infection. KhonKaen University S. Wongratanacheewin R. W. Sermswan S. Chareonsudjai Mahidol University S. Sirisinha S. Tangpradupkul P. Utaisincharoen

2. Melioidosis A great imitator with sign and symptom resemble many diseases Septicemia (dead in 1-3 days) High relapse rate DM is underlying dis. Wiersinga et al. 2006

3. Distribution of B. pseudomallei Clinical isolates:B. pseudomallei (Arabinose negative) Environemental isolates : B. thailandensis (Arabinose positive)

4. World wide distribution of Melioidosis Endemic in Thailand and Northern Australia 20 ºN, 20 ºS of the equator Cheng, A. C., and B. J. Currie. 2005. Clinical Microbiological Review. 18.2: 383 – 416

5. Epidemiology in Animal

6. Research Problems • Unknown pathogenesis mechanisms • What is (are) the main immunity that response to the bacteria? Innate>Adaptive? Vaccine? • High mortality and relapse rate? New regimens? • Various clinical profiles? • Rapid diagnostic? • Drug resistance may be exist. • Unknown environmental factors? • Human is an opportunistic host?

7. Our research objectives • การค้นหาองค์ประกอบของเชื้อ B. pseudomallei ที่เกี่ยวข้องกับการก่อให้เกิดพยาธิสภาพในโรคเมลิออยโดสิส โดยจะเน้นไปที่ LPS, capsule และ flagella • หายีนและกลุ่มยีนเป้าหมายอื่น เช่น Sigma N ที่มีผลและบทบาทต่อการก่อโรคของเชื้อ B. pseudomallei • ศึกษากลไกการป้องกันโรค และหาวิธีการขยายเวลาฉีด โดยใช้ CpGร่วมกับ liposome ในการป้องกันการติดเชื้อ B. pseudomallei

8. Phenotypes of bacteria used in this study 1026b SR117 Mutants were kindly provided by Professor Donald Woods, University of Calgary, Canada Wild type SR1015 SR117 MM35

9. 1.36x105 4.20x104 1.98x104 3.30x102 MOI 10:1 A549 Antibiotic protection assay • Invasion of non-phagocytic cell (A 549) Intracellular survival of mutants in non-phagocytic cell line A549

10. A 1026b SR1015 SR117 MM35 B 1026b SR117 SR1015 MM35 ความสามารถของเชื้อ mutants ที่จะทำให้เกิดพยาธิสภาพในเซลล์ A549 Percentage ของ MNGC (A) (9 ชั่วโมง) และใน (B) เป็น microscopic representation ของ cell fusion ในชั่วโมงที่ 7

11. Bacterial adherence in non-phagocytic cell line (A549) A549 4.5x105เซลล์ ถูก infect ด้วยเชื้อ B. pseudomalleiในอัตราส่วนแบคทีเรียต่อเซลล์ 10:1 แล้ว incubate ในตู้เลี้ยงเซลล์ที่อุณหภูมิ 37˚C ที่มี CO2 5% เป็นเวลา 1 ชั่วโมง

12. Internalization and replication ของเชื้อ mutants ในเซลล์มาโครฟาจของหนู (RAW 264.7) 1026b SR1015 SR117 MM35

13. Wikraiphat et al, 2009

14. A B Bacterial sensitivity to reactive nitrogen intermediates generated with GSNO (A) and SIN-1 (B)

15. 1026b SR1015 SR117 MM35 E.coli Serum bactericidal experiment (4 hrs)

16. iNOS U S.typhi 1026b SR1015 SRM117 MM35 Effect of reaction oxygen intermediate on mutants and wild type (using H2O2) • Inducible nitric oxide synthase (iNOS) production in phagocytic cell line (RAW 264.7)

18. A549 เซลล์ A549 ถูก infect ด้วยเชื้อในอัตราส่วน 10:1 ส่วน RAW 264.7 ถูก infect ในอัตราส่วนแบคทีเรียต่อเซลล์ 2:1 RAW • IκBα degradation and phosphorylation of p38 (pp38) in A549 and RAW 264.7

20. Cytokine production in phagocytic cell line (RAW 264.7)

23. Conclusion part I • LPS and capsule may associate with virulent of the bacteria. (in vivo). • LPS mutant (lack of O-polysaccharide but still have Lipid A and core antigen) lower invasion, adhesion, lower MNGC and susceptible to NHS, NO and antimicrobial peptide. • LPS mutant induce more cytokines (IL-1β, IL-12 and iNOS) than wild type • LPS and capsule may be involve in bacterial virulence.

24. Sigma N • The sigma factor 54 (RpoN) • The sigma factor 54 (RpoN) • The RpoN-dependent enhancer binding protein (EBP) • There are two sigma N found in Burkholderiapseudomalleilocate in two chromosome • Sigma N1 locates in chromosome 1, sigma N2 locates in chromosome 2

25. Genome sequence of B. pseudomallei • Sigma N 1 in Chromosome 1 : • Core function as cell growth, metabolism, macromolecule • synthesis, cofactor, chemotaxis and motility • Sigma N 2 in Chromosome 2: • Accessory functions as osmotic protection, regulation, secondary metabolism, adaptation and survival in different environments M. T. G. Holden., et al. PNAS2004; 39: 14240-14245

26. To study functions of sigma N in • Burkholderiapseudomalleiour approach • construction of B.p. sigma N mutants • both N1 and N2 • functional analysis of the mutant vs • its wild type • since N1 is involved with the core • function, therefore can’t construct • a mutant for sigma N1

27. rpoN2 5’ 3’ Bp. Wild type 1.5 kb I. Construction of B.p. rpoN 2 mutant Mya Myintzu Hluang; Thesis’ 2006

28. II. Construction of the B. pseudomallei rpoN2complement strain kb kb 2.0 1.7

29. III. Phenotypic characterization by amino acid utilization tests Bp. wild type amino acid utilization tests Bp. rpoN2 mutant Bp. rpoN2 complement

30. To examine the involvement of RpoN2 on amino acidutilization G: Growth, NG: Non Growth

31. Functional analysis involved in virulent factors • Multinucleated Giant Cell formation • Biofilm formation

32. Multinucleated Giant Cell formation Organization of TTSS cluster 3 on chromosome 2 Nichayapun, thesis,2005 Warawa et al: FEMS: Microbiol Lett 2005: 101-8

33. A. To identify TTSS genes underrpoN2 regulation 2 3 6 7 8 1 4 5 kb 0.5 574 0.3 398 180 0.1 • Lane 1 & 8 : 100 bp DNA Ladder marker • Lane 2 : PCR product of bsa Ztested in B. pseudomallei wild type • Lane 3 : PCR product of bsa Ztested in B. pseudomallei rpoN2 mutant • Lane 4 : PCR product of bipDtested in B. pseudomallei wild type • Lane 5: PCR product of bipDtested in B. pseudomallei rpoN2 mutant • Lane 6 : PCR product of bopAtested in B. pseudomallei wild type • Lane 7 : PCR product of bopAtested in B. pseudomallei rpoN2 mutant

34. Control without infectionWild Type Raw264.7 cell Wild Type infected with B.p. The mutant infected with B.p.

35. Biofilm formation CV staining -Result

36. CLSM-results in wild type b. Topro3 a. FITC-ConA c. Light microscope d. Merged image

37. CLSM-resultswild type vs sigma N2 mutant rpoN- 2.65 µm WT-3.75 µm

38. Conclusions for part II • We are not able to construct a B.p. sigma N1 mutant, since sigma N1 is involved in core functions. • We have successfully constructed a B.p. sigma N2 mutant and its complement strain. • The rpoN2 is involved in biosynthesis of amino acids: Ala, Ile, Cys, Met, and Asp • The rpoN2 also regulates some virulent properties: • Multinucleated Giant Cell formation • Biofilm formation

39. Wongratanacheewin, et al, 2004

40. CpG ทำให้หนูรอดชีวิตแต่ไม่สามารถกำจัดเชื้อให้หมดไปได้

41. Study of non ionic liposome with CpG in protection against B. pseudomallei in Balb/c mice

42. Macrophage in mice injected with CpG for 2 day enhanced bacterial uptake

43. Mice survived from CpG protection did not develop adaptive immunity

44. Mice survived from CpG protection did not develop adaptive immunity Protected mice Survival rate of 2 groups are not significant different Infected naïve mice

45. Include antigens in immunization procedure PP = Paraformaldehyde treated B. pseudomallei

46. G1: PP+CpG on day -30 G2: PP alone G3: PP day -30, CpG day 0 G4: Control (PBS)

47. Cationic liposome with CpG and antigens in immunization

49. When the challenge doses increase to 25LD50, the protection was lost.